Although powerful imaging systems exist for studying the morphologies of cells and subcellular architectures, no system currently exists to analyze the biochemical compositions of ultrasmall structures at the level of single copies. Despite the power of microscopy in providing nanoscale visual images, there remains a need to obtain chemical information about the subcellular compartments that are being visualized. To obtain information about the localization and function of complex cellular machineries, signaling pathways, and metabolic activities within the cell, a new system capable of providing comprehensive biochemical information about the micro- and nanometer-scale subcellular structures is required.
The primary reason for the lack of techniques in extracting chemical information from micro- and nano-scale subcellular structures lies in the minute amount of samples available for analysis. A typical single organelle may range in diameter from tens of nanometers to a couple micrometers, with a corresponding volume of approximately 6×10−20 L (e.g., for a 50-nm synaptic vesicle) to approximately 8×10−15 L (e.g., for a 2-μm mitochondrion). Within a volume of 6×10−20 L, even at a high concentration of 100 mM, the number of molecules present is only approximately 3600. At this small scale, most proteins would be present as a single copy or a few copies.
At the level of single cells, i.e., the entire contents of a cell rather than an organelle, a number of approaches have been proposed for single-cell microanalysis (Hyden, Trac-Trends Anal. Chem., 14: 141-148, 1995; Hyden, Trac-Trends Anal. Chem., 14: 148-154, 1995; Lowry, O. In METABOLISM OF THE NERVOUS SYSTEM; Richter, D.; Pergamon: London, page 325, 1952; Cannon, et al., Annu. Rev. Biophys. Biomolec. Struct., 29: 239-263, 2000), including voltammetric methods (Travis, et al., Annu. Rev. Biophys. Biomolec. Struct., 27: 77-103, 1998), separation-based strategies (Kennedy, et al., Science, 246: 57-63, 1989; Yeung, J. Chromatogr. A, 830: 243-262, 1999; Zhang, et al., Anal. Chem., 72: 318-322, 2000; Jankowski, et al., Trac-Trends Anal. Chem., 14: 170-176, 1995), and mass spectrometric techniques (Li, et al., Trends Biotechnol., 18: 151-160, 2000; Roddy, et al., Anal. Chem., 74: 4011-4019, 2002). In comparison with these single-cell studies, the chemical analysis of nanometer scale subcellular compartments presents formidable challenges. The sample volume available for analysis scales as the third power with the diameter of the cellular compartment. Therefore, a typical single mammalian cell with a diameter of approximately 10 μm has a volume of approximately 10−12 L, which is one thousand times the volume for a large subcellular structure of approximately 1 μm and ten million times the volume for a small synaptic vesicle with a diameter of approximately 50 nm. Most approaches used in single-cell studies are thus inadequate for nano-scale chemical analysis of subcellular structures.
The extremely small volume of the subcellular compartment, combined with a limited number of molecules in a complex mixture, necessitates a system that is both highly sensitive and capable of isolating each component for biochemical quantification and characterization. This invention describes a method and an integrated platform to perform this ultra sensitive analysis.